The Toll-like Receptors (TLRs) sense the presence of conserved microbial and host danger molecules and elicit primary inflammatory responses that mediate host defense. Excessive TLR signaling, however, often leads to inflammatory disease. Therefore, the ability to control TLR signaling is highly relevant to many pathologic conditions. TLR agonists induce the assembly of a multiprotein, intracellular signaling complex that mediates downstream signaling events. Assembly of this signaling platform is initiated by cooperative interactions of Toll-IL-1R resistance (TIR) domains present in TLR and TLR adapters. This study will identify and provide tools to target the key surface elements of TIR domains that mediate functional TIR-TIR interactions required for TLR2 and TLR4 signaling. Three Specific Aims are designed. The first Aim is to identify positions of the specific TIR interfaces that mediate functional TIR-TIR interactions. To achieve this Aim, for each TIR under study, we will generate a library of cell-permeable decoy peptides. Each peptide in a library will be composed of a cell-permeating sequence from Drosophila Antennapedia homeodomain in juxtaposition with a peptide of a TIR that represents a non-fragmented surface area of the TIR, so that each library encompasses the entire surface of the TIR. The peptides will be tested for their ability to inhibit TLR signaling in murine macrophages. The ability of a peptide to block signaling will be interpreted that this peptide represents a functional protein interface. The inhibitory peptides will be tested further in Aim 2 for the ability to bind directly to a TIR by the FRET approach so to confirm specificity of decoy-TIR interaction. Functionality of binary interactions identified by FRET will be further assessed by in silico analysis of the interface positions to identify interfaces that enable a TIR to interact simultaneously with several TIRs of the complex, thus establishing a cooperative, complex-stabilizing interaction. In Aim 3 we will determine therapeutic potential of targeting these key TIR elements for mitigation of TLR4 signaling elicited in mice by a sub-lethal or lethal LPS challenge. We anticipate that the proposed studies will identify the TIR molecular surfaces that mediate recruitment of adapters to TLR2 and TLR4 signaling complexes, and lay the groundwork for creation of novel anti-inflammatory therapeutics targeting the TLRs specifically. We also expect that the project will result in the development of a more generic approach for studying the formation of diverse signaling complexes.